Electroporation of normal human DNA endonucleases into xeroderma pigmentosum cells corrects their DNA repair defect

Cells from patients with the cancer-prone inherited disease, xeroderma pigmentosum (XP) are known to be defective in the endonuclease-mediated incision step in excision repair of a number of different types of DNA adducts, but the molecular events responsible have not been delineated. We have previously reported isolation of two DNA endonucleases, pI 4.6 and 7.6, from normal human chromatin which recognize adducts produced by psoralen plus long wavelength ultraviolet radiation (UVA). These endonucleases are both present in XP complementation group A (XPA) cells even though these cells are hypersensitive to this type of damage. We now report that introduction by electroporation of either normal endonuclease into XPA cells restored their markedly deficient DNA repair-related unscheduled DNA synthesis (UDS) to higher than normal levels following exposure to psoralen plus UVA. Introduction of XPA endonucleases into similarly treated XPA cells had little or no restorative effect on UDS. However, both normal and XPA endonucleases increased UDS in normal cells to higher than normal levels. These results indicate that XPA cells have endonucleases which can repair these adducts but which cannot function in intact cells unless a factor(s), which they lack is provided by normal cells.     

Base excision repair of DNA in {gamma}-irradiated human cells

Escherichia coli endonuclease IV was used to incise cellularDNA specifically at apurinic/apyrimidinic (AP) sites prior toalkaline elution to measure the resulting DNA strand breaks.-Irradiated HeLa cells initially contained DNA strand breaksand no AP sites. Upon incubation at 37?C the strand breaks wererapidly repaired and AP sites were generated and subsequentlyrepaired. The transient nature of the AP sites indicates thein vivo operation of a base excision repair pathway wherebydamaged bases are removed from DNA by DNA glycosylases to produceAP intermediates that are then substrates for AP endonucleases.     

A deficiency in a 230 kDa DNA repair protein in fanconi anemia complementation group A cells is corrected by the FANCA cDNA.

Cells from individuals with the cancer-prone, inherited disorder Fanconi anemia (FA) are hypersensitive to DNA interstrand cross-linking agents and this hypersensitivity correlates with a defect in ability to repair this type of damage to their DNA. We have isolated a DNA endonuclease complex from the nuclei of normal human cells which is involved in repair of DNA interstrand cross-links and have shown that in FA complementation group A (FA-A) cells there is a defect in ability of this complex to incise DNA containing interstrand cross-links. In order to identify the specific protein(s) in this complex which is defective in FA-A cells, monoclonal antibodies (mAbs) were developed against proteins in the normal complex. One of these mAbs, which is against a protein with a molecular weight of approximately 230 kDa, completely inhibited the ability of the normal complex to incise cross-linked DNA. Western blot analysis has shown that there is a deficiency in this protein in FA-A cells. Electophoretic analysis has also indicated that there are reduced levels of this protein in FA-A compared with normal cells. Studies carried out utilizing FA-A cells which have been stably transduced with a retroviral vector expressing the FANCA cDNA have shown that the DNA repair defect in these cells has been corrected; levels of unscheduled DNA synthesis are at least as great as those of normal human cells. In addition, in the transduced cells the deficiency in the 230 kDa protein has been corrected, as determined by both western blot and electrophoretic analysis. These results indicate that the FANCA gene plays a role in the expression or stability of the 230 kDa protein.     

Enhancement of Ca2+-dependent Endonuclease Activity in L1210 Cells during Apoptosis Induced by 1-β-D-Arabinofuranosylcytosine: Possible Involvement of Activating Factor(s)

Internucleosomal DNA fragmentation and morphological changes in nuclei typical of apoptosis were observed in L1210 cells incubated with 1.0 μg/ml of 1-β-D-arabinofuranosylcytosine (ara-C). To investigate the mechanisms involved, we examined the activities of endogenous endonucleases in nuclei and cytoplasm. Both fractions of control cells contained Ca²⁺ -dependent endonuclease which was capable of mediating internucleosomal DNA fragmentation. The assay system using two kinds of target substrates, i.e., nuclear chromatin of CCRF-CEM cells and naked DNA purified from the same cells, revealed that the activity of Ca²⁺-dependent endonuclease was enhanced in the crude nuclear extracts of cells treated with 1.0 μg/ml of ara-C for 24 h or 48 h. The activity was extracted more easily from ara-C-treated cells than control cells without sonication of the nuclear fraction. On the other hand, in the cytoplasmic fraction of the cells, the activity towards naked DNA was unchanged, whereas that towards nuclear chromatin was clearly enhanced. These results suggest that internucleosomal DNA fragmentation induced by ara-C treatment is associated with enhancement and activation of constitutively expressed Ca²⁺ -dependent endonuclease in L1210 cells.     

Anoxia-inducible endonuclease activity as a potential basis of the genomic instability of cancer cells.

Normal rat fibroblasts exhibit a staged response to anoxia which in several respects parallels processes activated in malignant tumor cells. We describe here a new element of the anoxic response, the induction by anoxia of a sequestered endonuclease activity. Such activity is elevated approximately 3-fold within anoxic fibroblasts and during Hirt DNA isolation is able to digest chromatin to produce a nucleosomal ladder. However, DNA is not measurably affected within intact cells, and cells retain complete viability as the endonuclease is induced. The anoxia-inducible endonuclease acts without specificity for DNA sequence. Trace leakage of this endonuclease into the nucleus has obvious potential to underlie the known propensity of anoxic cells to undergo amplification and may be associated with the break-related genomic instability of cancer cells.     

Visible light generates oxidative DNA base modifications in high excess of strand breaks in mammalian cells

The DNA damage induced by visible light in L1210 mouse leukaemiacells was analysed by an alkaline elution assay with specificrepair endonucleases. DNA single-strand breaks and DNA modificationssensitive to FPG protein (formamido-pyrimidine-DNA glycosylase),endonuclease III and exo-nuclease III were quantified in parallel.The light-induced cellular DNA damage was found to consist ofmany base modifications sensitive to FPG protein, which mostprobably are predominantly 7,8-dihydro-8-oxoguanine (8-hydroxy-guanine)residues. Base modifications sensitive to endonuclease III arevirtually absent. The yield of the FPG-sensitive base modificationsis 10-fold higher than that of single-strand breaks plus APsites (sites of base loss). The described ratios of the variousmodifications indicate that the damage most probably resultsfrom a reaction of DNA with singlet oxygen (type II reaction)or directly with an excited endogenous photosensitizer (typeI reaction) and is not mediated by hydroxyl radicals. Experimentswith cut-off filters indicate that wavelengths between 400 and500 nm are responsible for most of the modifications. The FPG-sensitivebase modifications are repaired efficiently (t     

Deficient gene specific repair of cisplatin-induced lesions in Xeroderma pigmentosum and Fanconi's anemia cell lines

Cisplatin is a chemotherapeutic agent known to cause DNA damage.The cytotoxicity of this drug is believed to result from theformation of DNA intrastrand adducts (IA) and DNA interstrandcrosslinks (ICL). While there are many studies on DNA repairof cisplatin damage at the overall level of the genome in varioushuman cell lines, there is little information on the gene-specificrepair. In this report, we have measured the formation and repairof cisplatin induced DNA adducts in the dihydrofolate reductase(DHFR) and ribosomal RNA (rRNA) genes in three cell lines: normalhuman fibroblasts, Fanconi's anemia complementation group A(FAA) and Xeroderma pigmentosum complementation group A (XPA).It is generally thought that XPA cells lack nucleotide excisionrepair and that FAA cells are deficient in the repair of DNAICL. We find that normal human fibroblast cells repair 84% ofthe ICL in the DHFR gene after 24 h, whereas XPA and FAA celllines only repaired 32 and 50% of the ICL respectively. Furthermore,69% of the cisplatin IA in the DHFR gene were repaired in 24h in normal human fibroblasts compared to 22% for XPA and 24%for FAA cells. The repair of the rRNA gene was less efficientthan in the DHFR gene, but the relative pattern between thedifferent cell lines was similar to that of the DHFR gene. Wethus find that FAA cells are deficient not only in the genespecific repair of cisplatin ICL, but also in the gene specificrepair of the more common cisplatin IA. XPA cells are normallythought to be without any nucleotide excision repair capacity,but our data could support a slight ICL unhooking activity.     

Enhanced S phase delay and inhibition of replication of an undamaged shuttle vector in UVC-irradiated xeroderma pigmentosum variant

Xeroderma pigmentosum variant (XP-V) cells are defective in bypass replication of UVC-induced thymine dimers in DNA because they lack a novel DNA polymerase (polymerase eta). In this study the effects of UVC on S phase cells were compared in fibroblasts derived from normal donors (IDH4) and XP-V patients (CTag) and immortalized by expression of the SV40 large T antigen. These transformed fibroblasts did not activate the G(1) checkpoint or inhibit replicon initiation when damaged by UVC or gamma-rays. The transformed XP-V cells (CTag) retained the increased sensitivity to UVC-induced inhibition of DNA strand growth previously observed with their diploid counterpart. Cell cycle progression analyses showed that CTag cells displayed a stronger S phase delay than transformed fibroblasts from normal individuals (IDH4) after treatment with only 2 J/m(2) UVC. Low doses of UVC also caused a lag in CTag cell proliferation. The extent of replication of an episomal DNA (pSV011), not previously exposed to radiation, was measured after the host cells were irradiated with 1-3 J/m(2) UVC. Replication of pSV011 was barely affected in irradiated IDH4 cells. Plasmid replication was inhibited by 50% in irradiated CTag cells and this inhibition could not be accounted for by increased killing of host cells by UVC. These results suggest that even in transformed cells UVC induces DNA damage responses that are reflected in transient cell cycle arrest, delay in proliferation and inhibition of episomal DNA replication. These responses are enhanced in CTag cells, presumably because of their bypass replication defect. The accumulation of replication complexes blocked at thymine dimers and extended single-stranded regions in chromosomal DNA might sequester replication factors that are needed for plasmid and chromosomal replication. Alternatively, aberrant replication structures might activate a signal transduction pathway that down-regulates DNA synthesis.     

Expression of a transfected DNA repair gene (XPA) in xeroderma pigmentosum group A cells restores normal DNA repair and mutagenesis of UV-treated plasmids

The XPA gene was initially cloned based on the ability of itscDNA to improve survival of cells from xeroderma pigmentosumcomplementation group A (XP-A) patients following irradiationof the cells with UV. We used plasmid host cell reactivationassays to compare UV mutagenesis and the proficiency of DNArepair in a cell line from an XP-A patient, XP2OS(SV40), twoderivative cell lines stably expressing XPA cDNAs and in a DNArepair proficient human cell line. Expression of XPA proteinin XP2OS cells allowed them to repair UV-treated plasmid pRSVCAT,increasing activity of the damaged CAT marker gene > 100-foldto levels produced by similarly damaged plasmids in normal cells.Expression of the XPA protein in XP2OS cells improved replicationof the UV-treated shuttle vector pSP189, increasing plasmidsurvival and decreasing plasmid mutation frequency to the levelsmeasured in normal cells. The sequence locations of most mutationhotspots in the plasmid marker gene were similar for the threecell lines and the differences did not correlate with the DNArepair status of the cells. This suggests that the locationof mutation hotspots is not directly influenced by DNA repair.Expression of the XPA protein did cause a shift in the typesof mutations seen in the plasmid gene. In the XP2OS cells >95%of the plasmid mutations were G: C     

Phosphorylation of nucleotide excision repair factor xeroderma pigmentosum group A by ataxia telangiectasia mutated and Rad3-related-dependent checkpoint pathway promotes cell survival in response to UV irradiation

DNA damage triggers complex cellular responses in eukaryotic cells, including initiation of DNA repair and activation of cell cycle checkpoints. In addition to inducing cell cycle arrest, checkpoint also has been suggested to modulate a variety of other cellular processes in response to DNA damage. In this study, we present evidence showing that the cellular function of xeroderma pigmentosum group A (XPA), a major nucleotide excision repair (NER) factor, could be modulated by checkpoint kinase ataxia-telangiectasia mutated and Rad3-related (ATR) in response to UV irradiation. We observed the apparent interaction and colocalization of XPA with ATR in response to UV irradiation. We showed that XPA was a substrate for in vitro phosphorylation by phosphatidylinositol-3-kinase-related kinase family kinases whereas in cells XPA was phosphorylated in an ATR-dependent manner and stimulated by UV irradiation. The Ser196 of XPA was identified as a biologically significant residue to be phosphorylated in vivo. The XPA-deficient cells complemented with XPA-S196A mutant, in which Ser196 was substituted with an alanine, displayed significantly higher UV sensitivity compared with the XPA cells complemented with wild-type XPA. Moreover, substitution of Ser196 with aspartic acid for mimicking the phosphorylation of XPA increased the cell survival to UV irradiation. Taken together, our results revealed a potential physical and functional link between NER and the ATR-dependent checkpoint pathway in human cells and suggested that the ATR checkpoint pathway could modulate the cellular activity of NER through phosphorylation of XPA at Ser196 on UV irradiation.     

DNA damage/repair studies using a combination of monoclonal thymidine antibody and unscheduled DNA synthesis simultaneously

The ability of a new monoclonal antibody against thymidine (MoAb 20B7) to detect chemotherapy induced single stranded DNA damage is described. HL-60 cells were used for these experiments. Damage to DNA was caused by incubation of cells with alkylating agents. Portions of DNA which is damaged are cleaved by cellular endonucleases, thus exposing thymidine on the opposite DNA strand. Processing of these samples by MoAb 20B7 showed that such damaged segments could be detected consistently. Furthermore, this method was combined with autoradiographic detection of unscheduled DNA synthesis thereby allowing for assessment of DNA repair simultaneously from the same cell.     

DNA damage binding protein component DDB1 participates in nucleotide excision repair through DDB2 DNA-binding and cullin 4A ubiquitin ligase activity

Functional defect in DNA damage binding (DDB) activity has a direct relationship to decreased nucleotide excision repair (NER) and increased susceptibility to cancer. DDB forms a complex with cullin 4A (Cul4A), which is now known to ubiquitylate DDB2, XPC, and histone H2A. However, the exact role of DDB1 in NER is unclear. In this study, we show that DDB1 knockdown in human cells impaired their ability to efficiently repair UV-induced cyclobutane pyrimidine dimers (CPD) but not 6-4 photoproducts (6-4PP). Extensive nuclear protein fractionation and chromatin association analysis revealed that upon irradiation, DDB1 protein is translocated from a loosely bound to a tightly bound in vivo chromatin fraction and the DDB1 translocation required the participation of functional DDB2 protein. DDB1 knockdown also affected the translocation of Cul4A component to the tightly bound form in UV-damaged chromatin in vivo as well as its recruitment to the locally damaged nuclear foci in situ. However, DDB1 knockdown had no effect on DNA damage binding capacity of DDB2. The data indicated that DDB2 can bind to damaged DNA in vivo as a monomer, whereas Cul4A recruitment to damage sites depends on the fully assembled complex. Our data also showed that DDB1 is required for the UV-induced DDB2 ubiquitylation and degradation. In summary, the results suggest that (a) DDB1 is critical for efficient NER of CPD; (b) DDB1 acts in bridging DDB2 and ubiquitin ligase Cul4A; and (c) DDB1 aids in recruiting the ubiquitin ligase activity to the damaged sites for successful commencement of lesion processing by NER.     

Downregulation of DNA excision repair by the hepatitis B virus-x protein occurs in p53-proficient and p53-deficient cells

Synergism between exposure to chemical carcinogens and infection with the hepatitis B virus (HBV) has been implicated in the high incidence of hepatocellular carcinoma. In this study we report that the HBV protein HBx, inhibits cellular DNA repair capacity in a p53-independent manner. Two alternative assays were used: the host cell reactivation assay, which measures the cell's capacity to repair DNA damage in a reporter plasmid, and unscheduled DNA synthesis, which measures the overall DNA repair capacity in damaged cells. Two p53-proficient cell lines, the hepatocellular carcinoma cell line HepG2 and liver epithelial cell line CCL13, were co-transfected with the pCMV-HBx reporter plasmid and the pCMV-CAT plasmid damaged with UVC radiation. Compared with cells transfected with control plasmid, the presence of HBx resulted in approximately 50% inhibition of the cell's capacity to reactivate CAT activity of UVC-damaged plasmid, and approximately 25% inhibition of unscheduled DNA synthesis in cells treated with either aflatoxin B1 epoxide or UVC radiation. Using the p53-deficient cell line Saos-2, we demonstrated that expression of HBx also resulted in diminished overall cellular DNA repair of damage induced by both aflatoxin B1 epoxide and UVC radiation, using both the host cell reactivation and unscheduled DNA synthesis assays. In summary, this study provides evidence for p53-independent regulation of DNA repair by HBx.     

Long-term XPC silencing reduces DNA double-strand break repair

To study the relationships between different DNA repair pathways, we established a set of clones in which one specific DNA repair gene was silenced using long-term RNA interference in HeLa cell line. We focus here on genes involved in either nucleotide excision repair (XPA and XPC) or nonhomologous end joining (NHEJ; DNA-PKcs and XRCC4). As expected, XPA(KD) (knock down) and XPC(KD) cells were highly sensitive to UVC. DNA-PKcs(KD) and XRCC4(KD) cells presented an increased sensitivity to various inducers of double-strand breaks (DSBs) and a 70% to 80% reduction of in vitro NHEJ activity. Long-term silencing of XPC gene expression led to an increased sensitivity to etoposide, a topoisomerase II inhibitor that creates DSBs through the progression of DNA replication forks. XPC(KD) cells also showed intolerance toward acute gamma-ray irradiation. We showed that XPC(KD) cells exhibited an altered spectrum of NHEJ products with decreased levels of intramolecular joined products. Moreover, in both XPC(KD) and DNA-PKcs(KD) cells, XRCC4 and ligase IV proteins were mobilized on damaged nuclear structures at lower doses of DSB inducer. In XPC-proficient cells, XPC protein was released from nuclear structures after induction of DSBs. By contrast, silencing of XPA gene expression did not have any effect on sensitivity to DSB or NHEJ. Our results suggest that XPC deficiency, certainly in combination with other genetic defects, may contribute to impair DSB repair.     

Inhibition of the human apurinic/apyrimidinic endonuclease (APE1) repair activity and sensitization of breast cancer cells to DNA alkylating agents with lucanthone

Cells repair DNA damage via four main mechanisms, however, damage induced by alkylators and oxidative damage is predominantly repaired by the DNA base excision repair (BER) pathway. The AP endonuclease, APE1, is one of the main enzymes in the BER pathway. It is abundant in human cells and accounts for nearly all of the abasic site cleavage activity observed in cellular extracts. APE1 expression is elevated in a variety of cancers and a high APE1 expression has been associated with poor outcome to chemoradiotherapy. The small molecule lucanthone has been shown to enhance the killing ability of ionizing radiation in cells and preliminary evidence suggests that lucanthone may inhibit AP endonuclease. Given the role APE1 plays in repairing oxidative and ionizing radiation DNA damage, the reports of lucanthone as an ionizing radiation enhancer and the potential use of lucanthone as an AP endonuclease inhibitor, we examined whether lucanthone could inhibit APE1 endonuclease activity. We report that lucanthone inhibits the repair activity of APE1, but not its redox function or exonuclease activity on mismatched nucleotides. Lucanthone also appears to inhibit exonuclease III family members (APE1 and ExoIII), but not endonuclease IV AP endonucleases, nor bifunctional glycosylase/lyases such as endonuclease VIII or formamidopyrimidine-DNA glycosylase (Fpg). Furthermore, the addition of lucanthone inhibits APE1 repair activity from cellular extracts and enhances the cell killing effect of the laboratory alkylating agent methyl methanesulfonate (MMS) and the clinically relevant agent temozolomide (TMZ). Given these initial findings, it would be of interest to further develop lucanthone as an APE1 inhibitor through the use of structure-function studies as a means of enhancing the sensitization of tumors to chemotherapeutic agents.     

Enhanced nucleotide excision repair capacity in lung cancer cells by preconditioning with DNA-damaging agents

The capacity of tumor cells for nucleotide excision repair (NER) is a major determinant of the efficacy of and resistance to DNA-damaging chemotherapeutics, such as cisplatin. Here, we demonstrate that using lesion-specific monoclonal antibodies, NER capacity is enhanced in human lung cancer cells after preconditioning with DNA-damaging agents. Preconditioning of cells with a nonlethal dose of UV radiation facilitated the kinetics of subsequent cisplatin repair and vice versa. Dual-incision assay confirmed that the enhanced NER capacity was sustained for 2 days. Checkpoint activation by ATR kinase and expression of NER factors were not altered significantly by the preconditioning, whereas association of XPA, the rate-limiting factor in NER, with chromatin was accelerated. In preconditioned cells, SIRT1 expression was increased, and this resulted in a decrease in acetylated XPA. Inhibition of SIRT1 abrogated the preconditioning-induced predominant XPA binding to DNA lesions. Taking these data together, we conclude that upregulated NER capacity in preconditioned lung cancer cells is caused partly by an increased level of SIRT1, which modulates XPA sensitivity to DNA damage. This study provides some insights into the molecular mechanism of chemoresistance through acquisition of enhanced DNA repair capacity in cancer cells.     

Increased error-prone NHEJ activity in myeloid leukemias is associated with DNA damage at sites that recruit key nonhomologous end-joining proteins

Double strand breaks (DSBs) are considered the most lethal form of DNA damage for eukaryotic cells, and misrepair of DSB can cause cell death, chromosome instability, and cancer. Nonhomologous end-joining (NHEJ) is a major mechanism for the repair of DSBs. We previously reported that the cancer predisposition Bloom's syndrome and myeloid leukemias demonstrate increased NHEJ activity and consequent misrepair. In this study, we link this increased NHEJ activity and infidelity to ongoing or induced DNA damage at sites that recruit key NHEJ proteins. We show here that in myeloid leukemia cells and normal hemopoietic cells, agents that induce DSBs produce an up to 2-fold increase in this DSB misrepair activity, whereas an alkylating agent produces little or no increases. Furthermore, NHEJ overactivity after induction of DSBs is dependent on the presence of Ku70/Ku86. We also present data to explain the constitutively activated NHEJ in myeloid leukemias. Using an immunofluorescence-based assay for DNA damage, myeloid leukemias demonstrate constitutive DNA damage in the absence of treatment with DSB-inducing agents compared with normal hemopoietic cells. Importantly, damaged foci from myeloid leukemia and normal cells colocalize with NHEJ proteins Ku70 and Ku86. These data suggest that the generation of increased constitutive DNA damage may be a common pathway for the creation of NHEJ-dependent genomic instability.     

Wavelength dependence of oxidative DNA damage induced by UV and visible light

DNA damage induced by UV radiation and visible light (290-500 nm) in AS52 Chinese hamster cells was analysed by an alkaline elution assay with specific repair endonucleases. Cells were exposed to extensively filtered monochrome or broad-band radiation. Between 290 and 315 nm, the ratio of base modifications sensitive to Fpg protein (i.e. 8- hydroxyguanine and formamidopyrimidines) and T4 endonuclease V (i.e. cyclobutane pyrimidine dimers) was constant (approximately 1:200), indicating that the direct excitation of DNA is responsible for both types of damage in this range of the spectrum. While the yield of pyrimidine dimers per unit dose continued to decrease exponentially beyond 315 nm, the yield of Fpg-sensitive modifications increased to a second maximum between 400 and 450 nm. The damage spectrum in this wavelength range consisted of only a few other modifications (strand breaks, abasic sites and pyrimidine modifications sensitive to endonuclease III) and is attributed to endogenous photosensitizers that give rise to oxidative DNA damage via singlet oxygen and/or type I reactions. The generation of Fpg-sensitive modifications by visible light was not linear with dose but followed a saturation curve. It is calculated that the exposure of the cells to low doses of solar radiation results in the formation of cyclobutane pyrimidine dimers and Fpg-sensitive modifications in a ratio of 10:1.      

A study of endonuclease III-sensitive sites in irradiated DNA: detection of alpha-particle-induced oxidative damage.

An important difference between chemical agents that induce oxidative damage in DNA and ionizing radiation is that radiation-induced damage is clustered locally on the DNA. Both modelling and experimental studies have predicted the importance of clustering of lesions induced by ionizing radiation and its dependence on radiation quality. With increasing linear energy transfer, it is predicted that complex lesions will be formed within 1-20 bp regions of the DNA. As well as strand breaks, these sites may contain multiple damaged bases. We have compared the yields of single strand breaks (ssb) and double strand breaks (dsb) along with those produced by treatment of irradiated DNA with the enzyme endonuclease III, which recognizes a number of oxidized pyrimidines in DNA and converts them to strand breaks. Plasmid DNA was irradiated under two different scavenging conditions to test the involvement of OH* radicals with either 60Co gamma-rays or alpha-particles from a 238Pu source. Under low scavenging conditions (10 mM Tris) gamma-irradiation induced 7.1 x 10(-7) ssb Gy/bp, which increased 3.7-fold to 2.6 x 10(-6) ssb Gy/bp with endo III treatment. In contrast the yields of dsb increased by 4.2-fold from 1.5 x 10(-8) to 6.3 x 10(-8) dsb Gy/bp. This equates to an additional 2.5% of the endo III-sensitive sites being converted to dsb on enzyme treatment. For alpha-particles this increased to 9%. Given that endo III sensitive sites may only constitute approximately 40% of the base lesions induced in DNA, this suggests that up to 6% of the ssb measured in X- and 22% in alpha-particle-irradiated DNA could have damaged bases associated with them contributing to lesion complexity.